Crosslinked polysaccharides obtained by covalent bonding of polysaccharide molecules are promising medical materials because of the high biocompatibility of polysaccharides per se and have been subjects of interest in various researches.
As the crosslinking methods, for example, the following four categories can be cited.
(1) Crosslinking with aldehyde crosslinking agents such as formaldehyde or glutaraldehyde.
(2) Self-crosslinking without any crosslinking groups which occurs between carboxyl groups and hydroxyl groups both present in the polysaccharides.
(3) Crosslinking with homo bifunctional crosslinking agents such as diepoxides, divinyl sulfones, diamines and dihydrazides, or hetero bifunctional crosslinking agents such as epihalohydrins.
(4) Crosslinking by the reaction of polysaccharides having functional groups such as photoreactive groups, polymerizable functional groups, amino groups, thiol groups and halogens, with polysaccharides having reactive groups which are complementary to the above functional groups.
Those methods in Category (1) have a safety problem. For example, Japanese Patent Kohyo Publication No. 2013-503688 describes that formalin or glutaraldehyde used as the crosslinking agent undergoes the crosslinking reaction also with proteins in a living body to give rise to tissue toxicity.
Regarding the self-crosslinking of Category (2), for example, take hyaluronic acid (HA) for instance, self-crosslinked ester-bonded hyaluronic acid wherein a portion or all of the carboxyl groups in a molecule are esterified with alcohol groups of the same polysaccharide molecule or of different polysaccharide molecules is disclosed in EP Patent No. 341745. Further, WO 99/10385 discloses self-crosslinked ester-bonded hyaluronic acid prepared by at least once freezing and subsequently thawing an aqueous hyaluronic acid solution under acidic conditions. WO 01/57093 discloses self-crosslinked ester-bonded hyaluronic acid prepared by mixing hyaluronic acid with an acidic solution so that the concentration will be not less than 5% and keeping the mixture as such without freezing the mixture. In methods in which an aqueous hyaluronic acid solution is frozen under acidic conditions, the dissolution half-life is increased as the freezing time is longer but excessively long freezing causes the molecular weight of the hyaluronic acid to be decreased, resulting in a shortening of the dissolution half-life. Further, the fact that the self-crosslinking occurs without any crosslinking agents makes it impossible to control the disintegration time by selecting an appropriate chemical structure of the crosslinking agent.
Regarding Category (3), Japanese Patent Kohyo Publication No. 2011-505362 discloses crosslinked products of hyaluronic acid or chondroitin sulfate (CS) obtained with diepoxide crosslinking agents such as 1,4-butanediol diglycidyl ether (BDDE).
Regarding Category (4), Adv. Mater., 2011, 23 (12), 1141-56 (Jason A. Burdick et al.) discloses the introduction of dihydrazide adipate, tyramide, methacrylate, glycidyl methacrylate, thiopropionyl hydrazide, bromoacetate and the like with respect to carboxyl groups and hydroxyl groups of hyaluronic acid.
As the example which aims at efficient modification of the physical properties of polysaccharides, a method to crosslink carboxyl groups of polysaccharides by diamines (corresponding to Category (3)) can be cited. For example, Japanese Patent Application Kokai Publication No. 2000-70356 discloses crosslinked products obtained by reacting carboxyl groups of polysaccharides such as alginic acid (Alg) and hyaluronic acid with alkyldiamines such as ethylenediamine. Further, Japanese Patent Kohyo Publication No. 2002-529549 discloses carboxymethyl cellulose (CMC) crosslinked with 1,3-diaminopropane. A report describes that chondroitin sulfate derivatives have been produced with use of diamines as the crosslinking agents (Biomaterials, 1995, 16, 473-478 (Amnon Sintov et al.)). However, various analyses of products prepared by reproducing the reported production method did not confirm the crosslinking of chondroitin sulfate (J. Biomater. appl., 1998, 12, 201-221 (C. Bourie et al.)) and concluded that the production of crosslinked chondroitin sulfate was unreal.
Some reports describe that esters are selected as biodegradable sites that allow crosslinked acidic polysaccharides to be disintegrated. For example, Adv. Mater., 2011, 23 (12), H41-56 (Jason A. Burdick et al.) reports that hyaluronic acid is crosslinked by the introduction of tyramide or glycidyl methacrylate followed by radical reaction or polymerization reaction. Further, Japanese Patent Application Kokai Publication No. H6-73102 presents a report in which a photosensitive compound is ester bonded to carboxyl groups in hyaluronic acid or chondroitin sulfate and the polysaccharide is photocrosslinked. These reports are associated with crosslinked products obtained by two-step reaction including the introduction of reactive groups and the crosslinking reaction.
2-Aminoethyl-2-aminoacetate (GlyC2N), illustrated in the right box below, is an example of the simplest structures of diamine crosslinking agents having a biodegradable site. According to Baldwin's rules (empirical rules describing the relative favourability of intramolecular nucleophilic reactions) (J. Chem. Soc., Chem. Commun. 1976, 734-736. (Jack E. Baldwin), J. Org. Chem., 1977, 42 (24), 3846-3852. (Jack E. Baldwin et al.)), the carbonyl group (an electrophilic group) in the ester bond and the amino group (a nucleophilic group) can form a 5-membered ring (5-exo-trig type). It is therefore understood that intramolecular nucleophilic substitution reaction is likely to occur in structures analogous to the above diamine (see below). In general, it is known that relatively simple exo-trig structures form a 3- to 7-membered ring in the transition state.

The left box above illustrates an example of Baldwin's rules. The right box illustrates the O→N acyl rearrangement of GlyC2N.
As a specific example of the relative favourability of intramolecular reactions, the similar structure -α-amino acid-(ester bond)-serine or threonine- is readily converted into -α-amino acid-(amide bond)-serine or threonine- by intramolecular nucleophilic reaction of the free amino group in serine to the carbonyl carbon in the ester bond (O→N acyl rearrangement), in water and under physiological conditions (Org. Lett., 2008, 10 (22), 5243-5246 (Natalia N. et al.), J. Org. Chem. 2006, 71, 6171-6177 (Irene Coin et al.)). S→N acyl rearrangement is also used in similar reactions (Science, 1994, 266 (5186), 776-779 (Dawson P. E. et al.)). These structures are generally called switch peptides or native chemical ligations and are frequently used as techniques for the chemical synthesis or structural conversion of peptides.
Based on the facts described above, it is conceivable that diamine crosslinking agents having a biodegradable site will easily undergo intramolecular nucleophilic substitution reactions, and actually there have been no reports describing that they are used as crosslinking agents for acidic polysaccharides.
Reports do exist which describe that compounds having the above diamine structure in part of the molecule are used for crosslinking reactions. For example, WO 96/34618 discloses a method in which 2-aminoethyl-2-aminoacetate and hexamethylene dicyanate are mixed with each other to form a polyfunctional crosslinking agent and proteins having amino groups are crosslinked therewith. Another example is reported in WO 99/11703 in which alginic acid, polyglutamic acid or carboxymethyl cellulose is crosslinked with a dihydrazide or a dicarboxylic acid wherein a 2-aminoglycinate skeleton is present in the dihydrazide skeleton or the dicarboxylic acid skeleton.
Even in these reports, the use of a crosslinker having such a structure as GlyC2N in the molecule involves the conversion of the amino structures at both ends into two carboxyl groups or two hydrazide groups to inhibit intramolecular reaction. That is, the crosslinking agents do not directly have a diamine structure with a biodegradable site in the molecule.
In spite of a variety of studies carried out on the crosslinking agents for acidic polysaccharides, there have been no reports of diamine crosslinking agents which, without combination with other kinds of molecules, allow for easy and simultaneous control of physical stress and disintegration time in accordance with use applications of medical materials while ensuring the high biocompatibility of acidic polysaccharides.